Late stage counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): Radioactivity-based biochemical assay to identify modulators of a panel of 48 kinases

Transcription factors are essential regulators of transcription that bind DNA to control both the rate and frequency of gene expression (1). Many diseases of cell homeostasis are associated with aberrant transcription factor activity (2). Colon cancer, in particular, is a disease of uncontrolled proliferation of the epithelial cells that line the intestinal crypts. Kruppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that binds to GC-rich sequences in promoters of numerous genes (3) including cyclin D1 (4), cyclin B1/Cdc2 (4), and integrin-linked kinase (5). KLF5 is highly expressed in rapidly dividing epithelial cells in intestinal crypts (6). This expression pattern of KLF5, along with studies demonstrating that KLF5 mediates the transforming effects of oncogenic H-Ras (7), and that ectopic expression of KLF5 leads to increased cell proliferation and anchorage-independent growth of cultured intestinal epithelial cells (8, 9), suggest that KLF5 may be involved in colon cancer pathogenesis. Therefore, the identification of selective inhibitors of KLF5 may provide useful tools to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium.

Assay Overview:The purpose of this assay is to determine whether powder samples of compounds identified as possible KLF5 inhibitor probe candidates change levels of KLF5 protein. This assay employs HT29 cells, which are human colorectal carcinoma cells. In this assay, HT29 cells are seeded in 12-well plates, treated with test compounds for 24 hours, followed by lysis in Laemmli buffer and determination of protein levels using standard western blotting protocols. Actin is used as an internal control. This assay was run in the assay provider's lab. Compounds were tested in singlicate at a nominal concentration of 10 uM.Protocol Summary:The parental HT29 cell lines were routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of McCoy's (HT29) supplemented with 10% v/v certified fetal bovine serum, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).Prior to the start of the assay 2 x 10e5 cells in a 1 mL volume of growth media were dispensed into each well of 12-well tissue culture-treated plates. The assay was started immediately by dispensing 4 mL of test compound in DMSO (0.4 % final DMSO concentration), DMSO alone to the appropriate wells. Next, the plates were incubated for 24 hours at 37 C (5% CO2, 95% RH. After 24 h of incubation, cells were lysed in 200 uL Laemmli buffer and subjected to electrophoresis in 10% polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane and developed with appropriate antibodies. The DMSO treated cells were considered as 100% (control). We did not perform any statistical analysis as this experiment was performed once.PubChem Activity Outcome and Score:Compounds were considered active if percent inhibition or activation in protein levels value were greater than or equal to 30%. Compounds were considered inactive if percent inhibition or activation in protein levels value were less than 30%.The reported PubChem Activity Score has been normalized to 100% observed absolute value of the fold change.The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 74-70.List of Reagents:Cells (assay provider);Laemmli buffer (assay provider)Primary antibodyRabbit anti-KLF5 (Millipore, Part 07-1580)Mouse anti-B-actin (Sigma Aldrich Part A1978)HRP-conjugated Secondary antibody: goat anti-rabbit and goat anti-mouse (vendor & part information not provided by assay provider)Chemiluminescent developer (Millipore, part not provided by assay provider)

This assay was run by the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.